We propose to use Drosophila as a model system for determining how neurotransmitter release at individual active zones is regulated. We will image synaptic vesicle fusion at single active zones to define how release probability, release mode (spontaneous versus evoked) and release plasticity is regulated at glutamatergic synapses. Neurotransmitters can be released through evoked fusion following an action potential, or by spontaneous fusion of vesicles (termed minis) in the absence of nerve stimulation. The two modes of vesicle release have been found at most synapses and are assumed to occur across the same population of active zones, though this has been difficult to define using classical approaches. A major technical limitation for the study of neurotransmitter release has been the inability to examine vesicle fusion at individual active zones. Electrophysiological studies of synaptic transmission measure the postsynaptic effect of neurotransmitter release over a large population of release sites, precluding an analysis of how individual active zones participate in and regulate synaptic vesicle fusion. We have developed transgenic tools that allow Ca2+ imaging of postsynaptic glutamate receptor activation following single vesicle fusion to spatially visualize all exocytotic events occurring through both spontaneous and evoked release pathways at Drosophila NMJs, allowing us to define general rules for vesicle fusion events at single active zones. Using these tools, we have begun to characterize the spatial and temporal dynamics of exocytotic events that occur spontaneously or in response to an action potential. We have also begun analyzing the relationship between these two modes of fusion at single release sites. Current data indicate a majority of active zones participate in both modes of fusion, although release probability is not correlated between the two modes of release and is highly variable across the population. Indeed, a subset of active zones is specifically dedicated to spontaneous release, indicating a population of postsynaptic receptors is uniquely activated by this mode of vesicle fusion. Using these new transgenic tools to visualize single active zone exocytosis, we will characterize how single release sites work, how they undergo plasticity, and how they contribute to both evoked and spontaneous fusion.